Recombinant lectin and uses thereof

ABSTRACT

Disclosed herein are a recombinant Streptomyces S27S5 hemagglutinin (SHA), and homologues thereof, and a fusion protein of a fluorescent protein (such as GFP and mCherry1) and SHA or a homologue thereof, which specifically bind to carbohydrates, including oligomeric sugars that terminate in L-rhamnose or D-galactose. The SHA, SHA homologues, and fusion proteins can be used to detect a variety of microorganisms or cancer or tumor antigens.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a divisional application of U.S. application Ser. No. 16/165,725, filed Oct. 19, 2018, which claims priority to U.S. Provisional Application Nos. 62/574,626 and 62/574,636, both filed on Oct. 19, 2017, the contents of which are incorporated herein by reference in their entireties, including the drawings.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant Number CA033572, awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing, which was submitted in ASCII format via EFS-Web, and is hereby incorporated by reference in its entirety. The ASCII copy, created on Aug. 4, 2021, is named SequenceListing.txt and is 38 KB in size.

BACKGROUND

In 1972, culture supernatants of 333 Actinomycetales bacterial strains isolated from the greater Tokyo area were screened for hem agglutination activity to identify microbial lectins (1,2). The culture supernatant of Streptomyces sp. 27S5 exhibited blood type B-specific activity. This was very unique at the time, because previously-known plant lectins were either A- or O-blood type-specific. Sixty mg of the identified lectin, named Streptomyces hemagglutinin (SHA), was purified to homogeneity from a 15-L culture broth of S. sp. 27S5 using gum arabic affinity chromatography, achieving a 13,300-fold enrichment with 64% recovery of the total activity (3). More than 200 mg of SHA was ultimately purified and subjected to various analyses. SHA was characterized as a small protein (˜11 kDa) with unique characteristics, such as rare blood type B specificity, an atypical tryptophan-rich nature, and two carbohydrate-binding sites (3,4). Accordingly, further study of SHA is needed.

SUMMARY

This disclosure relates to characterization, production of a recombinant SHA and homologues thereof and a fusion protein of a fluorescent protein and SHA that specifically bind to L-rhamnose or D-galactose, and novel uses of SHA and the fusion protein in detecting microorganisms that express carbohydrates containing L-rhamnose or D-galactose on the surface, or detecting tumor-expressed carbohydrates capable of specifically binding to SHA. Certain cancer/tumor cells expressing carbohydrates containing D-galactose on the surface can be detected by the methods disclosed herein. In some embodiments, the fusion protein is a fusion protein of a green fluorescent protein and SHA (GFP-SHA). In some embodiments, the fusion protein is a fusion protein of a red fluorescent protein and SHA (mCherry1-SHA). In some embodiments, the fusion protein is a non-aggregating protein. In some embodiments, the fusion protein is a soluble protein that is stable at about 4° C. for an extended period of time. In some embodiments, the fluorescent protein is linked to the N-terminus of SHA. In some embodiments, the fluorescent protein is linked to the C-terminus of SHA. In some embodiments, the fluorescent protein and SHA are linked via an acidic linker.

In one aspect, the disclosure provided herein relates to a recombinant Streptomyces S27S5 hemagglutinin (SHA), homologues thereof, and fusions proteins of a fluorescent protein (such as GFP or mCherry1) and SHA or homologues thereof (GFP-SHA fusion proteins or mCherry1-SHA fusion proteins). SHA, homologues thereof, SHA or homologues labeled with a marker such as a fluorescein or a derivative thereof, and GFP-SHA or mCherry1-SHA fusion proteins specifically bind to carbohydrates, including oligomeric sugars that terminate in L-rhamnose or D-galactose.

In another aspect, the disclosure provided herein relates to a method for detecting a microbial infection in a subject, wherein the microbial cell expresses a carbohydrate containing L-rhamnose or D-galactose on the surface. The method includes contacting a fluorescein or a derivative thereof labeled SHA or a fusion protein of a fluorescent protein and SHA disclosed herein with a sample from the subject, and detecting the fluorescence level in the sample, wherein the detection of fluorescence in the sample indicates the presence of the microbial infection. In some embodiments, the fusion protein is a fusion protein of a green fluorescent protein and SHA (GFP-SHA). In some embodiments, the fusion protein is a fusion protein of a red fluorescent protein and SHA (mCherry1-SHA). In some embodiments, the SHA is labeled with a fluorescein derivative such as fluorescein isothiocyanate (FITC). In some embodiments, the sample includes a biopsy sample, a tissue sample, a bronchoalveolar lavage sample, a blood sample, and a urine sample. In some embodiments, the microbial infection is caused by a bacterium or a fungus that expresses dTDP-4-dehydrorhamnose reductase gene (rmID). In some embodiments, the microbial infection includes mycoses caused by Candida albicans, Aspergillus fumigatus, or Fusarium solani. In some embodiments, the microbial infection is an infection by Streptococcus, Enterococcus or Lactococcus. In some embodiments, the microbial infection is invasive pulmonary aspergillosis, and the GFP-SHA fusion protein disclosed herein detects the presence of fungal galactomannan, indicating invasive pulmonary aspergillosis.

In another aspect, the disclosure provided herein relates to a method for detecting a cancer or tumor in a subject, wherein the cancer or tumor cell expresses a carbohydrate capable of specifically binding to SHA, a homologue thereof, a fragment of the SHA or a homologue thereof, a fluorescein or a derivative thereof labeled SHA, a homologue or fragment of the SHA, or a fusion protein of a fluorescent protein and SHA disclosed herein. In some embodiments, the cancer or tumor cell express a surface antigen containing D-galactose. In some embodiments, the method includes contacting a fluorescein or a derivative thereof labeled SHA, or a fusion protein of a fluorescent protein and SHA disclosed herein with a sample from the subject, and detecting the fluorescence level in the sample, wherein the detection of fluorescence in the sample indicates the presence of the cancer or tumor cell. In some embodiments, the sample includes a biopsy sample, a tissue sample, a bronchoalveolar lavage sample, a blood sample, and a urine sample. In some embodiments, the cancer includes colon cancer, pancreatic ductal carcinoma and pancreatic cancer. In some embodiments, the fusion protein is a fusion protein of a green fluorescent protein and SHA (GFP-SHA). In some embodiments, the fusion protein is a fusion protein of a red fluorescent protein and SHA (mCherry1-SHA). In some embodiments, the SHA is labeled with a fluorescein derivative such as fluorescein isothiocyanate (FITC).

In another aspect, the disclosure provided herein relates to a positron emission tomography (PET) probe comprising an SHA protein, a homologue thereof, a functional fragment of SHA or a homologue thereof, a fluorescein or a derivative thereof labeled SHA or a homologue or fragment of the SHA, or a fusion protein of a fluorescent protein and SHA disclosed herein labeled with a positron-emitting isotope. In some embodiments, the fusion protein is a fusion protein of a green fluorescent protein and SHA (GFP-SHA). In some embodiments, the fusion protein is a fusion protein of a red fluorescent protein and SHA (mCherry1-SHA). In some embodiments, the SHA is labeled with a fluorescein derivative such as fluorescein isothiocyanate (FITC).

In a related aspect, the disclosure relates to a method of imaging an organ or tissue having a microbial infection or detecting a location having a microbial infection caused by a microorganism expressing a carbohydrate containing L-rhamnose or D-galactose on the surface. The method entails administering to a subject suffering from or suspected of suffering from a microbial infection the PET probe described above, and imaging the organ or the tissue having the microbial infection by a PET scanning of the subject. Alternatively, the method entails administering to a subject suffering from or suspected of suffering from a microbial infection the PET probe described above, and detecting the location of the PET probe, thereby determining the location of the microbial infection. In some embodiments, the PET probe is locally administered to the subject. In some embodiments, the PET probe is systemically administered to the subject, e.g., by intravenous injection.

In yet another related aspect, the disclosure relates to a method of imaging a tumor or detecting a location having a cancer or tumor, where the cancer or tumor cell expresses an antigen comprising a carbohydrate capable of specifically binding to SHA, a homologue thereof, a fragment of the SHA and a homologue thereof, or a fluorescein labeled SHA or a homologue or fragment of the SHA. The method entails administering to a subject suffering from or suspected of suffering from a cancer or tumor the PET probe described above, and imaging the organ or the tissue having the cancer or tumor by a PET scanning of the subject. Alternatively, the method entails administering to a subject suffering from or suspected of suffering from a cancer or tumor the PET probe described above, and detecting the location of the PET probe, thereby determining the location of the cancer or tumor cells. In some embodiments, the PET probe is locally administered to the subject. In some embodiments, the PET probe is systemically administered to the subject, e.g., by intravenous injection.

BRIEF DESCRIPTION OF THE DRAWINGS

This application contains at least one drawing executed in color. Copies of this application with color drawing(s) will be provided by the Office upon request and payment of the necessary fees.

FIG. 1 illustrates the primer design for construction of rSHA.

FIG. 2 demonstrates SDS-PAGE analysis of archived SHA and thioredoxin (Trx) fused-SHA. S. sp. 27S5-produced SHA and a recombinant SHA homologue, Trx-SHA, were separated by SDS-PAGE on a 5-12% gradient gel and visualized with Coomassie Blue stain. Archived SHA (lane 1) was applied to a gum arabic gel column from which SHA was eluted by 1 M D-galactose (lane 2) or 0.2 M L-rhamnose (lane 3) in the presence of 1 M NaCl. Recombinant SHA was expressed in E. coli as a Trx-fusion protein and purified (lane 4). Gel image was assembled from three sections of the same gel; omitted spaces are indicated by the vertical lines.

FIG. 3 demonstrates the determination of the molecular mass of SHA. Electrospray Ionization (ESI) Fourier Transform Ion Cyclotron Resonance (FTICR)-MS of archived, forty-year old SHA revealed an average molecular mass of 13,314.67 Da, a monoisotopic mass of 13,306.65 Da, and the presence of a covalently attached hexose in ˜25% of the SHA molecules. The isotope distributions of the molecular SHA ions with a charge state of z=8 are magnified below the original spectrum.

FIG. 4 demonstrates LC MS/MS data of SHA proteolysis products aligned with the deduced amino acid sequence from the homologous partial sequence of the putative polysaccharide deacetylase of S. lavendulae (PDSL, WP 051840348.1). Overlapping SHA peptides were generated by separate enzymatic digestions and analyzed by high resolution Orbitrap LC-MS. Blue lines indicate database matches, grey lines mark matches through de nova sequencing with PEAKS software, and red-boxed 0 indicates methionine oxidation.

FIGS. 5A-5D show the primary structure of SHA. FIGS. 5A and 5B show that a single amino acid difference between recombinant SHA and the putative SHA domain of PDSL was identified by MALDI-MS of peptides from SHA and Trx-SHA, obtained after digestion with trypsin and LysC (5A) or ArgC (5B). FIG. 5C shows Orbitrap LC-MS of two ArgC-digested peptides of SHA before (non-reduced) and after (reduced) reduction with TCEP. FIG. 5 shows the sequence and primary structure of SHA derived from the results in FIGS. 5A-5C. Fragments identified by MS analyses (5A, 5B) are indicated by blue lines; S—S bonds identified by Orbitrap LC-MS (comparisons within 5C) are indicated by red linking lines; the A to E mutation at SHA position 108 (indicated by green labeled peak in 5A, and by blue linking lines in 5B) is highlighted in red.

FIGS. 6A-6C show glycan microarray analysis of archived and recombinant SHA. FIG. 6A shows representative heat maps of glycan-specific fluorescent signals (raw data in FIG. 7) in the absence (left panel) or in presence of 0.2 M L-rhamnose (right panel) using two different concentrations (1×, 0.1×) of SHA (top) or rSHA (bottom). FIG. 6B shows quantification of the normalized fluorescent glycan binding signals for archived and recombinant SHA, as in FIG. 6A (n=4). POS1-3 are positive controls containing standardized amounts of biotinylated IgGs; NEG are negative controls; numbers mark array positions of glycans with positive binding signals, as listed in FIG. 6B. Linker molecules are SP: OCH₂CH₂CH₂NH₂ and SP1: NH(CH₃)OCH₂CH₂NH₂. All other glycans that did not bind SHA/rSHA are listed in Table 4. FIG. 6C shows SDS-PAGE analysis of purified rSHA and archived SHA on a 4-12% gradient gel and visualized with Coomassie Blue staining.

FIG. 7 shows glycan microarray, original readouts from fluorescence scanner. Raw data of glycan-specific fluorescent signals in the absence (left panel) or presence of 0.2 M L-rhamnose (right panel) using two different concentrations (1×, 0.1×) of SHA (top) or rSHA (bottom). Yellow numbers indicate array positions with positive signals (see FIG. 6B for glycan identity).

FIGS. 8A-8C show SHA and homologues. FIG. 8A shows cross-species amino acid sequence comparison of SHA and the 11 closest SHA homologues. The boxes indicate SHA domains. Solid lines indicate experimentally defined disulfide bonds in SHA. FIG. 8B shows phylogenetic tree of SHA homologues (left). For percent sequence identity for proteins (center), number of identical residues/number of query residues matched are indicated in parentheses. Matching query length >131 indicates additional residues within the SHA homologues. For DNA (right), S. lavendulae DNA (438 bases) was used as the reference query for SHA homologues. Data are shown as percent identity/percent query covered of the corresponding nucleotide sequence. FIG. 8C shows comparison of the three SHA domains in the SHA protein.

FIG. 9 shows ¹H-NMR analysis for L-rhamnose binding to archived SHA. L-rhamnose was added to the SHA solution from 1 to 5 equivalents. The indole NH region (left) and methyl region (right) of 1D ¹H-NMR spectra are shown. Dashed red lines are included for alignment. Chemical shift changes observed for specific peaks indicate binding of L-rhamnose to SHA.

FIG. 10 shows representative fluorescent micrographs of Lactobacillus casei (Shirota) cells stained with recombinant GFP-SHA fusion protein (top left panel). Recombinant GFP was used as a negative control (top right panel). DAPI counterstaining is shown in blue in the merged micrographs (bottom).

FIG. 11 demonstrates ⁶⁸Ga PET imaging of CD-1 mice. These are the areas that contain microorganisms. While the control mouse only shows radioactivity uptake in the kidneys and in the bladder, which is typical for small proteins, the ⁶⁸Ga-DOTA-GFP-SHA-injected mouse reveals additional strong signals from the cecum and the small intestine. The latter are known internal organs that naturally contain a rhamnose-rich microbial flora.

FIG. 12 shows GFP-SHA staining of LS180 cell surfaces. Cells were stained with either 10 μg/mL GFP-SHA or a GFP-only control as indicated, followed by visualization using a Zeiss LSM 880 confocal microscope. DAPI was used as a counterstain to label nuclear DNA. GF=GFP mode; DAP=DAPI mode; Merge=GF+DP overlay.

FIG. 13 shows hematoxylin and eosin (H&E) and fluorescent staining of consecutive slices of cancerous and normal tissues from City of Hope patient. 40× magnification.

DETAILED DESCRIPTION

The following description of the invention is merely intended to illustrate various embodiments of the invention. As such, the specific modifications discussed are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein.

In one aspect, disclosed herein is a fusion protein, the amino acid sequence of which fusion protein comprising the amino acid sequence of a fluorescent protein and the amino acid sequence of SHA or an SHA homologue, and which fusion protein specifically binds to L-rhamnose or D-galactose. SHA or the SHA homologue specifically binds to L-rhamnose or D-galactose, and fusion to a fluorescent protein allows fluorescent detection of the SHA protein while retaining the specific binding to L-rhamnose or D-galactose. In some embodiments, the fluorescent protein includes GFP and mCherry1.

In some embodiments, SHA or the homologue thereof is a recombinant protein. In some embodiments, the amino acid sequence of SHA comprises three domains represented by SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, respectively. In some embodiments, each of the three domains comprises SEQ ID NO: 24 at the C-terminus. In some embodiments, the recombinant SHA (rSHA) has the following amino acid sequence:

(SEQ ID NO: 25) ARTVCYAAHVEGIGWQGAVCDGAVAGTTGQSRRMEAAVIATSGTGGVCAN AHLADIGWQGWACAADGKAVTVGTTGQSRRMEALGLQVGNGSVAAQAHVA DYGWLNAEGGNPVYVGTTGQSRRMEAVRIVVV. In some embodiments, the amino acid sequence of the SHA homologue is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 25. In some embodiments, the amino acid sequence of the SHA homologue is codon optimized.

In some embodiments, the fusion proteins encompassed in this disclosure include fusion proteins comprising GFP and a functional fragment of SEQ ID NO: 25 or a homologue thereof, as long as the functional fragment and the fusion protein of the GFP-SHA functional fragment are able to specifically bind to L-rhamnose and/or D-galactose. For example, a functional fragment of SEQ ID NO: 25 is a peptide homologous to a consecutive sequence of SEQ ID NO: 25 having substantially the same or even improved binding affinity to L-rhamnose and/or D-galactose comparing to the full length SHA protein represented by SEQ ID NO: 25. In some embodiments, the functional fragment is at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids, at least 110 amino acids, or at least 120 amino acids in length.

In some embodiments, the fusion proteins encompassed in this disclosure include fusion proteins comprising mCherry1 and a functional fragment of SEQ ID NO: 25 or a homologue thereof, as long as the functional fragment and the fusion protein of the mCherry1-SHA functional fragment are able to specifically bind to L-rhamnose and/or D-galactose. For example, a functional fragment of SEQ ID NO: 25 is a peptide homologous to a consecutive sequence of SEQ ID NO: 25 having substantially the same or even improved binding affinity to L-rhamnose and/or D-galactose comparing to the full length SHA protein represented by SEQ ID NO: 25. In some embodiments, the functional fragment is at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids, at least 110 amino acids, or at least 120 amino acids in length.

In some embodiments, mCherry1 and SHA are fused via an acidic linker with the sequences shown below. In some embodiments, the acidic linker increases solubility of the fusion proteins.

mCherry1-acidic-linker-SHA DNA sequence (SEQ ID NO: 26) (bases 1-708: mCherry1 (shown in capital letters); bases 709-735: acidic linker (shown in small letters, underlined); bases 736-1128: SHA (shown in capital letters, italic); bases 1129-1134: XhoI cloning site (shown in small letters, italic, underlined); bases 1135-1152: hexahistidine tag (shown in small letters); and bases 1153-1155: stop codon (shown in capital letters, bold):

¹ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCA TGCGCTTCAAGGIGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAG ATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAA GCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGT CCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGAC ATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCG CGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCT CCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAAC TTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACTATGGGCTGGGAGGC CTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCA AGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAG ACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGT CAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGG AACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAG CTGTACAAG⁷⁰⁹ ggtgacgaagtcgacgaagacgaaggt ⁷³⁶ GCGCGTAC CGTTTGCTACGCGGCGCACGTTGAAGGTATCGGTTGGCAGGGTGCGGTTT GCGACGGTGCGGTTGCGGGTACCACCGGTCAGTCTCGTCGTATGGAAGCG GCGGTTATCGCGACCTCTGGTACCGGTGGTGTTTGCGCGAACGCGCACCT GGCGGACATCGGTTGGCAGGGTTGGGCGTGCGCGGCGGACGGTAAAGCGG TTACCGTTGGTACCACCGGTCAGTCTCGTCGTATGGAAGCGCTGGGTCTG CAAGTTGGTAACGGTTCTGTTGCGGCGCAGGCGCACGTTGCGGACTACGG TTGGCTGAACGCGGAAGGTGGCAACCCGGTTTACGTTGGCACTACTGGTC AGTCCCGTCGTATGGAAGCGGTTCGTATCTGGGTT ¹¹²⁹ ctcgag ¹¹³⁵c accaccaccaccaccac¹¹⁵³ TGA

mCherry1-acidic-linker-SHA protein sequence (SEQ ID NO: 27) (AA 1-236: mCherry1; AA 237-245: acidic linker (italic, underlined); AA 246-378: SHA plus Leu-Glu from cloning site; and AA 379-384: hexahistidine tag):

  ¹Met-Val-Ser-Lys-Gly-Glu-Glu-Asp-Asn-Met-Ala- Ile-Ile-Lys-Glu-Phe-Met-Arg-Phe-Lys-Val-His-Met- Glu-Gly-Ser-Val-Asn-Gly-His-Glu-Phe-Glu-Ile-Glu- Gly-Glu-Gly-Glu-Gly-Arg-Pro-Tyr-Glu-Gly-Thr-Gln- Thr-Ala-Lys-Leu-Lys-Val-Thr-Lys-Gly-Gly-Pro-Leu- Pro-Phe-Ala-Trp-Asp-Ile-Leu-Ser-Pro-Gln-Phe-Met- Tyr-Gly-Ser-Lys-Ala-Tyr-Val-Lys-His-Pro-Ala-Asp- Ile-Pro-Asp-Tyr-Leu-Lys-Leu-Ser-Phe-Pro-Glu-Gly- Phe-Lys-Trp-Glu-Arg-Val-Met-Asn-Phe-Glu-Asp-Gly- Gly-Val-Val-Thr-Val-Thr-Gln-Asp-Ser-Ser-Leu-Gln- Asp-Gly-Glu-Phe-Ile-Tyr-Lys-Val-Lys-Leu-Arg-Gly- Thr-Asn-Phe-Pro-Ser-Asp-Gly-Pro-Val-Met-Gln-Lys- Lys-Thr-Met-Gly-Trp-Glu-Ala-Ser-Ser-Glu-Arg-Met- Tyr-Pro-Glu-Asp-Gly-Ala-Leu-Lys-Gly-Glu-Ile-Lys- Gln-Arg-Leu-Lys-Leu-Lys-Asp-Gly-Gly-His-Tyr-Asp- Ala-Glu-Val-Lys-Thr-Thr-Tyr-Lys-Ala-Lys-Lys-Pro- Val-Gln-Leu-Pro-Gly-Ala-Tyr-Asn-Val-Asn-Ile-Lys- Leu-Asp-Ile-Thr-Ser-His-Asn-Glu-Asp-Tyr-Thr-Ile- Val-Glu-Gln-Tyr-Glu-Arg-Ala-Glu-Gly-Arg-His-Ser- Thr-Gly-Gly-Met-Asp-Glu-Leu-Tyr-Lys-²³⁷ Gly-Asp- Glu-Val-Asp-Giu-Asp-G l u-G l y- ²⁴⁶Ala-Arg-Thr-Val- Cys-Tyr-Ala-Ala-His-Val-Glu-Gly-Ile-Gly-Trp-Gln- Gly-Ala-Val-Cys-Asp-Gly-Ala-Val-Ala-Gly-Thr-Thr- Gly-Gln-Ser-Arg-Arg-Met-Glu-Ala-Ala-Val-1le-Ala- Thr-Ser-Gly-Thr-Gly-Gly-Val-Cys-Ala-Asn-Ala-His- Leu-Ala-Asp-Ile-Gly-Trp-Gln-Gly-Trp-Ala-Cys-Ala- Ala-Asp-Gly-Lys-Ala-Val-Thr-Val-Gly-Thr-Thr-Gly- Gln-Ser-Arg-Arg-Met-Glu-Ala-Leu-Gly-Leu-Gln-Val- Gly-Asn-Gly-Ser-Val-Ala-Ala-Gln-Ala-His-Val-Ala- Asp-Tyr-Gly-Trp-Leu-Asn-Ala-Glu-Gly-Gly-Asn-Pro- Val-Tyr-Val-Gly-Thr-Thr-Gly-Gln-Ser-Arg-Arg-Met- Glu-Ala-Val-Arg-Ile-Trp-Val-Leu-Glu-³⁷⁹His-His- His-His-His-His

The SHA homologues having strong binding affinity to L-rhamnose or D-galactose are encompassed in this disclosure. These SHA homologues can be identified by their carbohydrate-binding properties, e.g., by using the commercially available Glycan Array 100 slides or other similar assays. Optionally, these SHA homologues can be further modified by substituting, deleting, or adding one or more amino acid residues to SEQ ID NO: 25. The modified SHA homologues may be tested for binding affinity to L-rhamnose and/or D-galactose to select the SHA homologues having similar or even improved binding affinity. Both SHA homologues without modification and the modified SHA homologues can be used for developing the GFP-SHA fusion proteins described above.

In some embodiments, SHA, the homologues or fragments of SHA can be labeled by a fluorescein or a derivative thereof such as FITC for easy detection. The fluorescein labeling has minimal impact on the binding activities of SHA or the homologue or fragment thereof.

In some embodiments, labeled or unlabeled SHA, SHA homologues or SHA fragments, and fusion proteins comprising a fluorescent protein and SHA or a homologue or fragment thereof disclosed herein, specifically bind to D-galactose and glycans containing Gal-α-1-3. In some embodiments, labeled or unlabeled SHA, SHA homologues or SHA fragments, and fusion proteins comprising a fluorescent protein and SHA or a homologue or fragment thereof disclosed herein, specifically bind to β-Gal-; α-Rha-; Gal-α-1,3-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-Gal-β-1,4-Glc-β-; Gal-α-1,4-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-(Fuc-α-1,2)-Gal-β- (for example, Blood B antigen trisaccharide); Gal-α-1,3-(Fuc-α-1,2)-Gal-β-1,4-Glc-β- (for example, Blood B antigen tetrasaccharide); or Gal-α-1,3-Gal-β-. In some embodiments, labeled or unlabeled SHA, SHA homologues or SHA fragments, and fusion proteins comprising a fluorescent protein and SHA or a homologue or fragment thereof disclosed herein, specifically bind to Gal-α-1,4-Gal-β-1,4-Glc-β-; GaINAc-β-1,3-Gal-β-1,4-Glc-β-; or Gal-α-1,4-Gal-β-1,4-GlcNAc-β-. In some embodiments, labeled or unlabeled SHA, SHA homologues or SHA fragments, and fusion proteins comprising a fluorescent protein and SHA or a homologue or fragment thereof disclosed herein, specifically bind to a carbohydrate terminating in or to a polysaccharide having one or more branches terminating in β-Gal-; α-Rha-; Gal-α-1,3-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-Gal-β-1,4-Glc-β-; Gal-α-1,4-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-(Fuc-α-1,2)-Gal-β-; Gal-α-1,3-(Fuc-α-1,2)-Gal-β-1,4-Glc-β-; Gal-α-1,3-Gal-β-, Gal-α-1,4-Gal-β-1,4-Glc-β-; GaINAc-β-1,3-Gal-β-1,4-Glc-β-; or Gal-α-1,4-Gal-β-1,4-GlcNAc-β-. In some embodiments, the fluorescent proteins include GFP and mCherry1. In some embodiments, the fluorescein or a derivative thereof includes FITC.

This disclosure demonstrates that archived SHA produced by S. sp. 27S5 and purified 40 years ago remained intact and maintained its carbohydrate-binding and hemagglutination (data not shown) activities, and that the molecular mass and primary structure of the archived SHA were successfully determined using modern mass spectrometric/proteomic strategies. The amino acid sequence of SHA was partially determined by Edman degradation methods in the 1970s, as described in the thesis of YFY(7). That study found redundancy in the N-terminal amino acid sequences of BrCN-cleaved SHA peptides, which was reasoned to be due to the presence of microheterogeneity in the purified protein. The primary structure disclosed herein clearly reveals that the difficulty of sequencing SHA in the 1970s was due to the three homologous SHA domains, which occupy 70% of the SHA molecule. It is fortuitous that the putative SHA gene of S. lavendulae was found in the Streptomyces genome database, which was expanded within two months after this protein was first revisited after 40 years. Consequently, the primary structure of SHA was revealed at last.

FTICR-MS revealed an average molecular mass of 13,314.67 Da and the presence of a covalently attached hexose in ˜25% of the SHA molecules. The MS results suggest that hexose may be a component of SHA. Glycation of Lys in macromolecules, including hemoglobin, serum albumin, crystalline, and collagens, has been well studied (12-14). Given that the original SHA was obtained from a culture medium containing 2% D-fructose, it is possible that D-fructose was non-enzymatically attached to ε-amino groups of Lys. SHA was exposed to a high concentration of D-galactose after the original affinity purification, and significant amounts of D-galactose were found remaining in the archived SHA sample. Thus, it is also possible that D-galactose present in the SHA solution may have caused such a covalent linkage. Alternatively, it is possible that the hexose was added post-translationally by Streptomyces. However, the mass spectrometric data indicated multiple hexose-modified residues and not a single defined site (data not shown), hinting the presence of an inhomogeneous chemical reaction rather than a well-defined in vivo posttranslational modification.

The 131-amino acid primary structure of SHA was solved by showing that peptides derived from SHA aligned to the C-terminal two-thirds of the putative protein from S. lavendulae with >99% identity. Close comparison of peptides derived from SHA and the SHA domain of the putative protein revealed a single amino acid substitution at the SHA-equivalent position 108 in the putative protein, from E to A. Recombinant SHA(A108E) showed the same carbohydrate-binding specificity and similar affinity for L-rhamnose as archived SHA. These results confirmed that SHA is identical to the N-terminally truncated hypothetical protein in the genome of S. lavendulae, except that, in the putative protein, E in SHA-position 108 is substituted by A. The SHA(A108E) gene was used to express SHA proteins in different forms, including GFP-SHA. After the confirmation of L-rhamnose and D-galactose glycan specificity of the SHA(A108E) protein, this protein was designated as rSHA.

As the working examples demonstrate, SHA and eleven hypothetical protein homologues have three ChW-like SHA domains. To date, ChW domains have been exclusively found in the C. acetobutylicum species. The three ChW-like domains identified in SHA and its homologues represent additional examples for non-C. acetobutylicum proteins containing ChW domain repeats. The ChW domain is 45-47 amino acids long, and features an absolutely conserved tryptophan and high contents of hydrophobic and small amino acids. SHA homologues contain five conserved tryptophan residues, four of which are located in the three ChW-like SHA domains. Like the three SHA domains in SHA, the ChW domains cluster into groups of threes, which suggests they function as a triplet (10). Although carbohydrate recognition functions have been suggested (9), no conclusive study has been published as to the role of ChW domains.

The identified tryptophan residues may be involved in the binding function of SHA. It was previously reported that the circular dichroism (CD) spectrum of SHA strongly resembled that of poly(L-tryptophan), and speculated that tryptophan side chains contributed to a positive CD band at 226 nm (3). It was also suggested a potential involvement of tryptophan residues in L-rhamnose binding to SHA (4). Those conclusions were based on solvent-perturbation studies, which demonstrated that the number of solvent-exposed Trp (or average extent of exposure) was two in the absence of L-rhamnose, and three in the presence of L-rhamnose. This suggested that one tryptophan residue appears outside as a result of SHA binding to this sugar. Oxidation of two tryptophan residues with N-bromosuccinimide led to complete loss of its carbohydrate-binding activity, which also indicated that these tryptophan residues are important for retaining this activity (4). Using NMR, the current study confirmed the involvement of tryptophans in the binding of SHA to L-rhamnose. In analogy, another L-rhamnose-specific protein, α-L-rhamosidase of S. avermitilis has three tryptophan residues binding to L-rhamnose via hydrophobic interaction to the pyranose ring of the sugar (15).

The above-mentioned structural information is helpful for understanding specificity and affinity of SHA. It is important to carry out extensive binding assays of SHA against a variety of glycans. In this study, the specific binding of both archived SHA and rSHA was compared side by side, using the Glycan Array 100, and at two concentrations of SHA in the absence and presence of L-rhamnose. Although semi-quantitative, the results clearly revealed the following: (1) SHA bound to D-galactose and glycans containing Gal-α-1-3, which is the key signature of blood type B specificity, as well as L-rhamnose; (2) SHA bound to L-rhamnose with the highest affinity among glycans tested, as evidenced by the fact that the binding to L-rhamnose was still observed when other positive binding signals were abolished in the presence of 0.2M L-rhamnose; (3) SHA and rSHA showed the same glycan specificity profile, suggesting that rSHA represents the authentic SHA. These results are consistent with those previously published (2-4), confirming the blood type B and L-rhamnose specific nature of SHA. Gum arabic has been effectively used to purify SHA and SHA fusion proteins in the past and this study. As previously reported (2), hemagglutination of type B-erythrocytes by SHA was inhibited in the presence of plant-originated galactomannans, with guar gum>locust bean gum>gum arabic. The glycan structure resembling guar gum, locust bean gum, and gum arabic remains to be determined. These galactomannans are used in foods as stabilizers, and it is interesting to note that in the clinical setting, fungal galactomannan is used as a biomarker for invasive pulmonary aspergillosis, a life-threatening infection mainly affecting immunocompromised patients (16).

Although microbial lectins with similar characteristics to SHA have not been reported, significant data on L-rhamnose binding lectins (RBLs) from fish eggs are available (17-21). Interestingly, RBLs from a number of different fish species are composed of two or three domains consisting of approximately 100 amino acids, which are known as carbohydrate-recognition domains (RBL CRDs) (22,23). A lectin purified from sea urchin (Anthocidaris crassispina) eggs (SUEL) was reported to contain a galactose-binding lectin domain (24), but was later shown to bind to L-rhamnose preferentially, which seems reasonable given that L-rhamnose and D-galactose share the same hydroxyl group orientation at C2 and C4 of the pyranose ring structure (22,23). The RBL CRD, also called SUEL-type lectin domain, is composed of eight highly conserved half-Cys and several other conserved segments, e.g., YGA in the N-terminal and DP and K in the C-terminal domain (22). However, RBL CRD shows no homology to SHA domains, due to its domain size, which is over three times longer than the SHA domain; the absence of tryptophan, which is the signature of SHA domains; and its heavily disulfide-linked domain structure.

The functions of L-rhamnose-specific lectins are of particular interest. One suggested physiological role of fish egg lectins is as a defense mechanism against pathogenic bacteria (17). Rhamnose-binding lectins from salmon and trout are involved in innate immunity and recognition of lipopolysaccharides (LPS) or lipoteichoic acid (LTA), respectively, on the cell surface of bacteria (20,25). In contrast to animal lectins, lectins produced by microorganisms have different functions. Bacterial surface agglutinins with mannose specificity play roles in cell-cell interactions, as well as in microbial pathogenicity (26). The related functions of SHA are expected to include interactions with outside cells, such as attaching to neighboring plants and surrounding microorganisms, in addition to potential defense mechanisms. The closest SHA homologue was found in the S. lavendulae genome encoding a putative protein. If expressed by Streptomyces, this enzyme would be expected to catalyze the N- or O-deacetylation of acetylated sugars on the membranes of Gram-positive bacteria. However, it is not likely that SHA has such deacetylation activities, as SHA does not seem to recognize N-acetylated carbohydrates, as seen in the glycan array results.

The comparison of SHA to genomically-derived hypothetical proteins revealed the intriguing observation that the SHA-homologous domains of all eleven hypothetical proteins are localized in the C-terminal regions of the larger ORFs. Under the culturing conditions described (3), expression of the SHA-homologous proteins encoded by the genomes of S. lavendulae and S. sp. Mg1 was not observed (data not shown). In contrast, when the original study was performed in the 1970s, three HA activity-positive strains were identified from the 333 Actinomycetales culture supernatants screened (1). During the original screening, culture supernatants were serially diluted and incubated with blood type A, B, O, or AB erythrocyte suspensions. Supernatants that showed HA activity at 4- or 8-fold dilutions on titer plates were considered to be substantially positive; this included S. sp. 27S5 (1). SHA was purified from culture supernatants of S. sp. 27S5 by gum arabic affinity chromatography (3). It is possible that SHA could have been expressed as a precursor protein with an unknown N-terminal sequence, a signal sequence, and a protease-processing site, so that SHA molecules could be found in the culture broth, as observed 40 years ago.

As disclosed herein, the recombinant GFP-SHA binds to L. casei Shirota cells. Additional bacteria and fungi can be screened to identify microorganisms that interact with SHA. A similar approach was reported for a recombinant horseshoe crab plasma lectin that recognizes specific pathogen-associated molecular patterns of bacteria through L-rhamnose (27).

The SHA protein and homologues thereof, as well as fusion proteins of GFP and an SHA protein, an SHA homologue, or a functional fragment thereof (GFP-SHA and mCherry1-SHA fusion proteins) have a variety of novel uses in detecting the presence of certain microorganisms or cancer or tumor cells, diagnosing certain microbial infections or certain types of cancer or tumor, and detecting or imaging the location of microbial infections, or cancer or tumor, e.g., by PET scanning. Ideally, the fusion proteins are soluble, non-aggregating and stable for an extended period of time. The fusion proteins also retain the binding activity of SHA, a homologue or fragment thereof. The fusion proteins can bind well to the gum-Arabic carbohydrate column material and can be eluted with L-rhamnose or D-galactose.

For example, various bacteria or fungi expressing dTDP-4-dehydrorhamnose reductase gene (rmID) may be detected in vitro by contacting the bacteria or fungi with a GFP-SHA or mCherry1-SHA fusion protein disclosed herein and monitoring the presence or change of fluorescence. In some embodiments, the bacterial or fungal cell expresses a carbohydrate containing L-rhamnose or D-galactose and display the carbohydrate on the surface of the cell. The GFP-SHA and mCherry1-SHA fusion proteins can detect the presence of such microorganisms in various liquid samples from a subject, e.g., a biopsy sample, a tissue sample, a bronchoalveolar lavage sample, a blood sample, and a urine sample. Alternatively, if the bacterial or fungal cell expresses a carbohydrate containing L-rhamnose or D-galactose but does not display the carbohydrate on the surface of the cell, the GFP-SHA and mCherry1-SHA fusion proteins can detect the presence of such microorganisms in fixed tissue samples, e.g., paraffin-fixed or formalin-fixed and paraffin-embedded tissue samples.

Similarly, various cancer or tumor cells expressing tumor-specific carbohydrates can be detected using the GFP-SHA and mCherry1-SHA fusion proteins disclosed herein. Such carbohydrates contain or terminate in beta-Galactose- (β-Gal-); Gal-α-1,3-; Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-Gal-β-1,4-Glc-β-; Gal-α-1,4-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-(Fuc-α-1,2)-Gal-β-; Gal-α-1,3-(Fuc-α-1,2)-Gal-β-1,4-Glc-β-; Gal-α-1,3-Gal-β-; Gal-α-1,4-Gal-β-1,4-Glc-β-; GaINAc-β-1,3-; Gal-β-1,4-Glc-β-; or Gal-α-1,4-Gal-β-1,4-GlcNAc-β-. In some embodiments, the core structures of the Thomsen Friedenreich and mucin antigens terminate in galactose and therefore, can be detected by the GFP-SHA and mCherry1-SHA fusion proteins disclosed herein. The GFP-SHA and mCherry1-SHA fusion proteins can detect the presence of such cancer or tumor cells in various liquid samples from a subject, e.g., a biopsy sample, a tissue sample, a bronchoalveolar lavage sample, a blood sample, and a urine sample. Alternatively, if the cancer or tumor cell expresses a carbohydrate capable of specifically binding to SHA, a homologue thereof, or a fragment of the SHA or a homologue thereof disclosed herein but does not display the carbohydrate on the surface of the cell, the GFP-SHA and mCherry1-SHA fusion proteins can detect the presence of such a cancer or tumor cell in fixed tissue samples, e.g., paraffin-fixed or formalin-fixed and paraffin-embedded tissue samples. In some embodiments, the cancer is colon cancer.

The GFP-SHA and mCherry1-SHA fusion proteins can be used for diagnosis of various microbial infections caused by one or more microorganisms expressing a carbohydrate containing L-rhamnose or D-galactose or various cancers or tumors expressing tumor-specific carbohydrates capable of specifically binding to SHA, a homologue thereof, or a fragment of the SHA or a homologue thereof disclosed herein. The method entails the step of contacting a sample obtained from a subject suffering from a microbial infection or a cancer or tumor with a GFP-SHA fusion protein or an mCherry1-SHA fusion protein, and determining the fluorescence level in the sample, wherein the presence of the fluorescence indicating the condition of the microbial infection or cancer or tumor. In some embodiments, the method further entails the step of contacting a sample obtained from a negative control subject, such as a healthy subject or the subject before the microbial infection or without cancer or tumor, with a GFP-SHA or mCherry1-SHA fusion protein, and comparing the fluorescence levels of the sample of the negative control subject with the sample of the subject suffering from the microbial infection or cancer or tumor, wherein the difference in the fluorescence levels indicating the microbial infection or presence of cancer or tumor. Alternatively, the fluorescence level of a negative control subject can be established by an average or median fluorescence level of a population of healthy subjects who do not suffer from the microbial infection or cancer or tumor. In some embodiments, the cancer is colon cancer.

In a related aspect, this disclosure relates to a method of determining the prognosis of treating a microbial infection caused by one or more microorganisms expressing a carbohydrate containing L-rhamnose or D-galactose or a cancer or tumor expressing a carbohydrate tumor antigen capable of specifically binding to SHA, a homologue thereof, or a fragment of the SHA or a homologue thereof disclosed herein. The method entails the step of contacting a sample obtained from a subject suffering from a microbial infection or a cancer or tumor with a GFP-SHA or mCherry1-SHA fusion protein to determine the fluorescence level, treating the subject suffering from a microbial infection with one or more antimicrobial agents or the subject suffering from a cancer or tumor with one or more cancer therapies, contacting a sample obtained from the subject after the treatment with a GFP-SHA or mCherry1-SHA fusion protein to determine the fluorescence level, and comparing the fluorescence levels before and after the treatment to determine the prognosis of the treatment. The method can further comprise administering to the subject an alternative antimicrobial agent or cancer therapy or an additional amount of the antimicrobial agent or cancer therapy if a desired prognosis is not achieved. In some embodiments, the cancer is colon cancer.

Infections caused by various microorganisms or certain types of cancers or tumors can be detected based on the specific binding of the GFP-SHA or mCherry1-SHA fusion proteins disclosed herein with the carbohydrate containing L-rhamnose or D-galactose displayed on the surface of the microorganisms or the carbohydrate tumor antigen capable of specifically binding to SHA, a homologue thereof, or a fragment of the SHA or a homologue thereof disclosed herein. As demonstrated in the working examples, the GFP-SHA or mCherry1-SHA fusion proteins and fluorescein labeled SHA disclosed herein can specifically bind to β-Gal-; α-Rha-; Gal-α-1,3-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-Gal-β-1,4-Glc-β-; Gal-α-1,4-Ga-β-1,3-GlcNAc-β-; Gal-α-1,3-(Fuc-α-1,2)-Gal-β-; Gal-α-1,3-(Fuc-α-1,2)-Gal-β-1,4-Glc-β-; Gal-α-1,3-Gal-β-, Gal-α-1,4-Gal-β-1,4-Glc-β-; GalNAc-β-1,3-Gal-β-1,4-Glc-β-; or Gal-α-1,4-Gal-β-1,4-GlcNAc-β-. The GFP-SHA fusion proteins can be used to detect microbial infections caused by a microorganism expressing a carbohydrate terminating in or otherwise exposing the aforementioned monosaccharides or oligosaccharides. Alternatively, the GFP-SHA and mCherry1-SHA fusion proteins can be used to detect cancer or tumor cells expressing a carbohydrate tumor antigen terminating in or otherwise exposing the aforementioned monosaccharides or oligosaccharides. In some embodiments, the cancer is colon cancer.

In yet another related aspect, disclosed herein is a method of imaging a local microbial infection site caused by a microorganism expressing a carbohydrate containing L-rhamnose or D-galactose. Alternatively, the method of imaging a tumor site, where the tumor cell expresses a tumor-specific antigen including a carbohydrate capable of specifically binding to SHA, a homologue thereof, or a fragment of the SHA or a homologue thereof disclosed herein, can be performed in a similar way. In some embodiments, the cancer is colon cancer. A GFP-SHA or mCherry1-SHA fusion protein, labeled or unlabeled SHA protein or a homologue thereof, or a functional fragment of the SHA protein or a homologue thereof, can be labeled with a PET isotope to produce a PET probe. The method entails the step of administering to a subject suffering from a microbial infection or a cancer or tumor the PET probe, and performing a PET scanning of the subject to image the location of the microbial infection or cancer or tumor, or to detect the location of the PET probe, thereby determining the location of the microbial infection or the cancer or tumor. In some embodiments, the PET probe is administered to the subject by intravenous injection. In some embodiments, the PET probe is locally administered to the microbial infection site or the tumor site.

The selection and use of a suitable PET probe can be done based on the knowledge in the field (29). For example, the PET imaging can be performed using a DOTA-labeled GFP-SHA or mCherry1-SHA fusion protein. DOTA is a chelator (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) that is used to covalently attach PET imaging metal isotopes to proteins. For example, ⁶⁸Ga or ⁶⁴Cu can be used in this technique. There are many other PET metal isotopes that can be used and are compatible with DOTA chelation. Other labeling techniques also can be used, for example, non-metal PET isotopes including 124-Iodine, 18F, etc.

In some embodiments, the DOTA labeling is conducted via an attachment to amino acids such as lysine (via amino groups) or cysteines (via thiols). In some embodiments, the DOTA labeling is attached to one or more amino acids located in the SHA protein, a homologue thereof, or a functional fragment of the SHA protein or a homologue thereof. In some embodiments, the DOTA labeling is attached to one or more amino acids located in GFP or mCherry1 of a GFP-SHA or mCherry1-SHA fusion protein.

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

EXAMPLES Materials and Methods

Materials-S. lavendulae strain NCIB 6959/ATCC 14158 and HEK293S cells were purchased from ATCC (Manassas, Va.). S. sp. strain Mg1 was a kind gift from Dr. Paul Straight of Texas A&M University (6). E. coli C41(DE3) and E. Clone® were from Lucigen (Middleton, Wis.). Gum arabic was purchased from Sigma-Aldrich (St. Louis, Mo.). MS-grade Trypsin, LysC, ArgC, V8 protease, and pepsin were from Promega (Madison, Wis.). Chymotrypsin was from Worthington Biochemical (Lakewood, N.J.). pET32b and pcDNA3.1 vectors were from Merck Millipore (Billerica, Mass.) and Thermo Fisher Scientific (Waltham, Mass.), respectively.

Purification and characterization-SHA was purified forty years ago as described (3) and kept frozen at −80° C. The purity and quality of the archived SHA were determined using SDS-PAGE. The N-terminal amino acid sequence of SHA was determined using Edman degradation performed on the Procise 494HT Protein Sequencing System (Applied Biosystems, Thermo Fisher Scientific).

Specific binding of SHA to gum arabic gels-Gum arabic gels were prepared according to published methods (3). The archived SHA as well as recombinant SHA proteins were applied to the gum arabic gel column. After washing the column, SHAs were eluted with either 1 M D-galactose in the presence of 1 M NaCl as described (3), or 0.2 M L-rhamnose in the presence of 1 M NaCl.

NMR titration study-NMR analysis was performed using a DRX-500 spectrometer equipped with a cryogenic TXI probe (Bruker BioSpin, Billerica, Mass.). The probe temperature was set to 298 K. Archived SHA (0.1 mg) was dissolved in 500 μl of 20 mM sodium phosphate buffer, pH 6.5 (H₂O:D₂O=9:1). L-rhamnose solution was added to the SHA solution at molar ratios from 1:0 to 1:5 (SHA:L-rhamnose). Data processing and analysis were performed using XWIN-NMR (ver. 3.5, Bruker BioSpin). NMR spectra were displayed with XWIN-PLOT (ver. 3.5, Bruker BioSpin).

Mass Spectrometry—To determine molecular mass, the intact archived SHA was analyzed using electrospray ionization (ESI) Fourier Transform Ion Cyclotron Resonance (FTICR)-MS on a Thermo LTQ FTICR (Thermo Fisher) at ˜500,000 resolution.

To determine the amino acid sequence of SHA, overlapping SHA peptides were obtained by performing separate enzymatic digestions with trypsin, chymotrypsin, LysC, ArgC, V8 protease, and pepsin, and analyzed by LC-MS on an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific, Waltham Mass.), as well as by MALDI-MS on a SimulTof Combo 200 instrument (SimulTOF Systems, Virgin Instruments, Marlborough, Mass.). MS and MS/MS collision-induced dissociation (CID) fragmentation data from these peptides were analyzed with Xcalibur software (Thermo Fisher Scientific) and with PEAKS Studio software (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada).

SHA disulfide bond determination was made using MALDI-MS and high (120,000) resolution Thermo Orbitrap Fusion Tribrid Mass Spectrometer analysis of the intact protein and the digested protein, before and after reduction with 50 μM TCEP, pH 2.0, at 80° C. for 30 min.

Expression of an SHA homologous recombinant protein—the SHA homologous domain of the putative protein from S. lavendulae was expressed, which showed the highest homology to SHA (>99% identity), as a recombinant protein. To develop this recombinant SHA homologue, a synthetic gene expressing a wild-type SHA of the putative protein and a mutant SHA gene with an A to E amino acid substitution at position 108 (A108E), were produced using E. coli codon-optimized overlapping oligo DNA primers, and cloned into pET32b (Table 1). The primer binding sites are illustrated in FIG. 1.

TABLE 1 Primer SEQ Name ID NO: DNA Sequence P1f 1 TGCGCGAACGCGCACCTGGCGGACATCGGTTGGCAGGGTTGGGCGTGCGCGGCGGACGGT P2f 2 CGTATGGAAGCGGCGGTTATCGCGACCTCTGGTACCGGTGGTGTTTGCGCGAACGCGCAC P3f 3 GTTTGCGACGGTGCGGTTGCGGGTACCACCGGTCAGTCTCGTCGTATGGAAGCGGCGGTT P4f 4 GCGGCGCACGTTGAAGGTATCGGTTGGCAGGGTGCGGTTTGCGACGGTGCGGTTGCGGGT 5af 5 AAAGAATTCGCGCCGGCGGCGCGTACCGTTTGCTACGCGGCGCACGTTGAAGGTATCGGT P1r 6 TCCATACGACGAGACTGACCGGTGGTACCAACGGTAACCGCTTTACCGTCCGCCGCGCAC P2r 7 GCCTGCGCCGCAACAGAACCGTTACCAACTTGCAGACCCAGCGCTTCCATACGACGAGAC P3r 8 TTACCACCCGCCGCGTTCAGCCAACCGTAGTCCGCAACGTGCGCCTGCGCCGCAACAGAA P4ar 9 CGACGGGACTGACCAGTAGTGCCAACGTAAACCGGGTTGCCACCCGCCGCGTTCAGCCAA P5ar 10 TTTCTCGAGTTAAACCCAGATACGAACCGCTTCCATACGACGGGACTGACCAGTAGTGCC PDSL 11 GACTACGGTTGGCTGAACGCGGAAGGTGGCAACCCGGTTTACGTTGGC A108Ef PDSL 12 GCCAACGTAAACCGGGTTGCCACCTTCCGCGTTCAGCCAACCGTAGTC A108Er

The recombinant wild-type SHA was expressed in E. coli C41(DE3) as a thioredoxin (Trx) fusion protein with His-tag. Trx-SHA was purified from E. coli cell pellets derived from a 2-L culture by solubilization and affinity purification on a Ni-NTA resin (Thermo Fisher Scientific). The purified wild-type SHA was digested with multiple enzyme combinations, as described above for SHA, to compare resulting peptides from both proteins.

Due to solubility issues various fusion proteins of the recombinant SHA were prepared and expressed in E. coli. Of those, a yeast SUMO(SMT3)-fusion protein was successfully purified for comparing carbohydrate-binding specificity with that of archived SHA. Briefly, SMT3-fused SHA(A108E) was prepared by insertion at the SMT3 and Ulp1 cleavage sites of pET32b/SHA(A108E). E. Cloni® (Lucigen) was transformed by pET32b/SMT3-SHA(A108E). SMT3-SHA(A108E) was purified using a His6-tag specific nickel-NTA column from transformed cells after solubilization with 5M urea/B-Per lysis buffer (Pierce), followed by refolding in the presence of 1 M galactose and 10 mM β-mercaptoethanol. SHA(A108E) was cleaved off from SMT3 bound to the column by incubating with UPL1. The resulting SHA(A108E) was purified by gum arabic gels. The authenticity of SHA(A108E) was confirmed by SDS-PAGE and glycan microarray analyses.

Glycan microarray analyses—Microarray analysis was performed according to the manufacturer recommendations using RayBio Glycan Array 100 (RayBiotech, Norcross Ga.) slides. Each slide contains four sub-microarrays printed with 100 synthetic glycans. Briefly, 200 μL of 0.1 mg/mL of both archived SHA and rSHA were dialyzed overnight at 4° C. against 1×PBS dialysis buffer to avoid contaminating samples with amines prior to biotinylation. Dialyzed samples were incubated with biotin-containing reaction solution at 22° C. for 30 min. Sub-arrays were blocked for 30 min at 22° C. After biotinylated SHA samples were diluted with 1×PBS, 400 μL of each sample was added to each sub-array. Slide #1 sub-arrays were incubated with 400 μL of 20 μg/mL (1×) or 2 μg/mL (0.1×) SHA, in the absence or presence of 0.2 M L-Rhamnose. Slide #2 sub-arrays were incubated with 400 μL of 20 μg/mL (1×) or 2 μg/mL of rSHA (0.1×), in the absence or presence of 0.2 M L-rhamnose. Slides were incubated for 16 h at 4° C. for highest intensities. Washing was performed according to the manufacturer's protocol, followed by incubation with Cy3 dye-conjugated streptavidin. The slides were incubated at 22° C. for 1 h with gentle shaking, then washed multiple times as recommended. The signals were visualized using an Agilent DNA microarray scanner (Model G2505C; Agilent, Santa Clara, Calif.) at 532 nm for Cy3. Data extraction and analysis was performed after subtraction of the background and normalization to the internal references provided by the manufacturer, using an ImageJ Protein Array Analyzer software (28).

Staining of Lactobacillus casei (Shirota) cells by fluorescently labeled SHA-Recombinant GFP-SHA was expressed by inserting the SHA(A108E) gene at the C-terminus of GFP in pET28/GFP, followed by transformation of E. cloni cells. GFP-SHA was purified from cell pellets collected from 4-L culture, after solubilization with 5 M urea/B-Per lysis buffer (Pierce), using a His6-tag specific nickel-NTA column, followed by refolding in the presence of 1 M galactose and 10 mM β-mercaptoethanol, and eluting with 400 mM imidazole. GFP-SHA was concentrated using Centricon YM10 centrifugal filters (Fisher Scientific) and purified by FPLC with Superdex 75G (GE Healthcare Life Science, Pittsburgh, Pa.).

L. casei Shirota cells were isolated from commercially available Yakult yogurt drink. The authenticity of L. casei Shirota was verified by Sanger sequencing of its 16S rRNA by showing 100% match to the reference sequence AB531131. Four hundred ml Difco™ Lactobacilli MRS Broth (Fisher Scientific) was inoculated with L. casei Shirota cells at a concentration of 10⁶ cells/mL. L. casei Shirota cells were grown for 16 h at 37° C., harvested by centrifugation, and washed three times with 1×PBS. Cells were re-suspended in 5 ml 70% ethanol and incubated at 22° C. for 30 min under continuous rotation. Cells were washed three times with 1×PBS, then re-suspended in 5 ml 1×PBS. Bacterial cells were blocked for non-specific binding with 3% BSA in PBS and NP-40 (0.5%) for 30 min, followed by 1 h incubation with 50 μM of GFP-SHA or GFP as a negative control. Cells were washed three times with 1×PBS and finally re-suspended in 1 mL PBS containing 10% glycerol. Bacteria cells were counter-stained with DAPI (3 μM) and examined using a Zeiss Observer II system (Carl Zeiss, Jena, Germany). Fluorescent images were analyzed using Image-Pro Plus and ZEISS ZEN software (Carl Zeiss, Jena, Germany).

Example 1: Preservation of Active SHA

It was first confirmed that the SHA protein that was purified 40 years ago and archived in a frozen state, was intact, readily bound to a gum arabic affinity chromatography column, and specifically eluted with a competing monosaccharide D-galactose or L-rhamnose as shown in FIG. 2, lanes 1-3.

Example 2: Determination of the Molecular Mass of SHA Using Mass Spectrometry

The molecular mass of SHA was previously estimated to be approximately 11 kDa, based on various approaches, including gel filtration in the presence of 6 M guanidine hydrochloride, SDS-PAGE, and sedimentation equilibrium analysis (3). Electrospray ionization (ESI) Fourier Transform Ion Cyclotron Resonance (FTICR) MS was applied to determine the molecular mass of SHA more precisely. This high-resolution mass spectrometric technique revealed a precise average molecular mass of 13,314.67 Da, a monoisotopic mass of 13,306.65 Da, and the presence of a covalently attached hexose in ˜25% of the SHA molecules (FIG. 3).

Example 3: Identification of SHA Homologues in Streptomyces Genomes

To determine the sequence identity of SHA, bottom-up proteomics experiments were conducted. SHA was digested separately with several proteases to generate overlapping peptides. These peptides were then analyzed by liquid chromatography (LC) coupled with high-resolution multistage mass spectrometry (MS/MS). An initial database search was performed and revealed a closely matching SHA homologue in the genome of Streptomyces sp. Mg1 as a hypothetical protein (GenBank accession #EDX26679.1) (6); data not shown. More Streptomyces genome sequences became available later; subsequently, a more refined search led to the identification of a homologue in S. lavendulae with even better scores for MS/MS database matching. The digested SHA peptides aligned almost completely with the deduced C-terminal amino acid sequence of the putative polysaccharide deacetylase of S. lavendulae (Accession number WP_051840348.1; FIG. 4). SHA matched with the C-terminal 131 amino acids of the hypothetical 199-amino acid protein, except for a partial sequence stretch consisting of nine amino acids from SHA-position 101-109. However, the mass spectrometric data did not cover any sequence of the N-terminal portion of either the S. sp. Mg1 or S. lavendulae protein, comprising 74 and 68 amino acid residues, respectively.

Example 4: Determination of the N-Terminal Amino Acid Sequence of SHA

Previous amino acid sequencing of reduced and carboxymethylated SHA revealed the N-terminal amino acids to be AxTVCYAAxV (SEQ ID NO: 13) (7); x indicates an undetermined residue. To confirm these results and identify additional amino acids in the sequence, N-terminal sequencing of the archived SHA was performed. Approximately 30 amino acids were identified to be ARTVcYAAHVEGIGWQGAVcDGAVAxTtxQsRr (SEQ ID NO; 14) (lowercase letters indicate tentative identification). Together, the two independent N-terminal sequencing results strongly suggested that the N-terminal sequence of SHA was ARTVCY (SEQ ID NO: 15).

Example 5: Solution of the Primary Structure of SHA

By considering the N-terminal sequencing information, the molecular mass of the intact SHA protein, and the database matching with digested peptides, the SHA sequence appeared to be almost identical to the C-terminal portion of the putative protein, residues 69-199. To identify how the amino acid sequences differ between SHA and the SHA domain of the putative protein, a recombinant thioredoxin (Trx)-SHA fusion protein was generated and peptide fingerprints of the recombinant SHA and SHA putative protein were compared. First, the homologous SHA domain from the putative protein was cloned into a PET32 vector to transform E. coli, from which the fusion protein was purified using Ni-NTA resin (FIG. 2, lane 4). Then the purified recombinant Trx-SHA fusion protein was digested using multiple enzymes to generate overlapping peptides for LC-MS and MALDI-MS analyses, as for SHA above. Finally, the LC-MS/MS data sets of the digested peptides from the recombinant SHA and its homologue SHA putative protein were compared. It was found that the sequence of the recombinant SHA differed from that of SHA putative protein by a single A108E change (FIGS. 5A and 5B). This was confirmed by calculating and comparing the molecular masses for the recombinant SHA and SHA putative protein as a 58-Da mass difference.

To determine disulfide bonds in SHA, endoproteinase ArgC was used to digest archived SHA, with or without reduction with tris(2-carboxyethyl) phosphine (TCEP), followed by high resolution Orbitrap LC-MS. Comparison of the spectra of two digested peptides before and after TCEP reduction showed a clear 2-Da mass difference (FIG. 5C). This indicates that SHA contains two consecutive disulfide bonds that connect cysteine residues C5 with C20 and C48 with C63, as illustrated in FIG. 5D. No other disulfide bond-connected peptides were detected. Taken together, these results allowed deduction of the primary structure of SHA (illustrated in FIG. 5D, and summarized in Tables 2 and 3, along with repetitive domain structures).

Table 2 demonstrates that three homologous SHA domains, consisting of 92 amino acids, form 70% of the total amino acids of SHA. The primary sequence of SHA is principally composed of three homologous SHA domains 1, 2, and 3, consisting of 29, 33, and 30 amino acids, respectively. Together, the three SHA domains comprise 92 amino acids, 70% of the total 131 amino acids in SHA. Underlining indicates the completely matched 11-amino acid sequences in these domains. Homology among the three SHA domains is shown in FIG. 81.

TABLE 2 Number ChW-  of  like Location Amino acid sequence residues domains  1-7 ARTVCYA (SEQ ID NO: 16) 7  8-36 AHVEGIGWQGAVCDGAVAGTTGQ 29  SHA  SRRMEA (SEQ ID NO: 17)  domain 1 37-50 AVIATSGTGGVCAN (SEQ ID 14  NO: 18) 51-83 AHLADIGWQGWACAADGKAVTVG 33 SHA  TTGQSRRMEA (SEQ ID NO: domain 2  19) 84-96 LGLQVGNGSVAAQ (SEQ ID 13  NO: 20)  97-126 AHVADYGWLNAEGGNPVYVGTTG 30  SHA  QSRRMEA (SEQ ID NO: 21)  domain 3 127-131 VRIWV (SEQ ID NO: 22) 5 

Table 3 demonstrates the homology between ChW and SHA domains. SHA domains 1, 2, and 3 were compared to Clostridium acetobutylicum ATCC 824 protein Q97E41, which was found using SMART (simple modular architecture research tool). The key signature of ChW domains, tryptophan (W), is underlined.

TABLE 3 ChW AHVQNIGWQDWVSNGAEAGTDGKGLRVEA Identity domain LRIKLENMP (SEQ ID NO: 23) SHA AHVEGIGWQGAVCDGAVAGTTGQSRRMEA 17/29 = domain 1 (SEQ ID NO: 17) 59% SHA AHLADIGWQGWACAADGKAVTVGTTGQSR 13/33 = domain 2 RMEA (SEQ ID NO: 19) 39% SHA AHVADYGWLNAEGGNPVYVGTTGQSRRME 11/30 = domain 3 A (SEQ ID NO: 21) 37%

Example 6: Characterization of the Carbohydrate-Binding Properties of SHA

In contrast to archived SHA, the first Trx-SHA fusion protein generated as described above was poorly soluble and not suitable for functional analyses. Therefore, a novel construct that encoded an E. coli codon-optimized, His-tagged, Trx-SMT3 (SUMO family protein)-SHA(A108E) fusion protein was expressed. This protein was refolded on the nickel NTA column in the presence of D-galactose, and soluble recombinant SHA (rSHA) was cleaved off from the Trx-SMT3 portion using His-tagged Ulp1 (8). The purified rSHA is shown in FIG. 6C, together with archived SHA. To determine the carbohydrate-binding specificity of SHA and rSHA, the commercially available Glycan Array 100 slides were used, on which 100 synthetic glycans of the most frequently identified structures in the literature are mounted in each of four sub-arrays. The glycan binding was measured using two distinct concentrations of biotin-labeled SHAs (1×: 20 μg/ml and 0.1×: 2 μg/ml) in the absence and presence of 0.2 M L-rhamnose as a competitive inhibitor. Both SHA and rSHA showed identical carbohydrate-binding specificities (FIGS. 6A, 6B, and 7). Strong signals were observed for SHA/rSHA binding to: β-Gal-; α-Rha-; Gal-α-1,3-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-Gal-β-1,4-Glc-β-; Gal-α-1,4-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-(Fuc-α-1,2)-Gal-β-[Blood B antigen trisaccharide]; Gal-α-1,3-(Fuc-α-1,2)-Gal-β-1,4-Glc-β- [Blood B antigen tetrasaccharide]; and Gal-α-1,3-Gal-β-. Also, weaker, but still significant, binding to Gal-α-1,4-Gal-β-1,4-Glc-β-; GaINAc-β-1,3-Gal-β-1,4-Glc-β-; and Gal-α-1,4-Gal-β-1,4-GlcNAc-β- was observed. SHA/rSHA did not bind to the remaining 89 chip-immobilized glycans (Table 4).

TABLE 4 Array Position Glycan 1 β-Glc-Sp 3 α-Man-Sp 4 α-Fuc-Sp 6 β-GlcNAc-Sp 7 β-GalNAc-Sp 8 Tobramycin 9 Gal-β-1,3-GlcNAc-β-Sp 11 Neu5Ac-α-2,3-Gal-β-1,3-GlcNAc-β-Sp 12 Neu5Ac-α-2,6-Gal-β-1,3-GlcNAc-β-Sp 13 Neu5Gc-α-2,3-Gal-β-1,3-GlcNAc-β-Sp 14 Neu5Gc-α-2,6-Gal-β-1,3-GlcNAc-β-Sp 15 Gal-β-1,3-(Fuc-α-1,4)-GlcNAc-β-[Lewis A]-Sp 16 Gal-β-1,4-Glc-β-Sp 19 GlcNAc-β-1,3-Gal-β-1,4-Glc-β-Sp 21 Neu5Ac-α-2,3-Gal-β-1,4-Glc-β-Sp 22 Neu5Ac-α-2,6-Gal-β-1,4-Glc-β-Sp 23 Neu5Gc-α-2,3-Gal-β-1,4-Glc-β-Sp 24 Neu5Ac-α-2,6-Gal-β-1,4-Glc-β-Sp 25 Gal-β-1,4-(Fuc-α-1,3)-Glc-β-Sp 26 GalNAc-β-1,3-Gal-α-1,4-Gal-β-1,4-Glc-β-Sp 27 GlcNAc-β-1,6-GlcNAc-β-Sp 28 4-P-GlcNAc-b-1,4-Man-b-Sp 29 Glc-α-1,2-Gal-α-1,3-Glc-α-Sp 30 Gal-β-1,3-GalNAc-α-Sp 31 Gal-β-1,4-GlcNAc-β-Sp 32 Gal-β-1,4-(Fuc-α-1,3)-GlcNAc-β-[Lewis X]-Sp 33 Neu5Ac-α-2,3-Gal-β-1,4-(Fuc-α-1,3)-GlcNAc-β-[Sialyl Lewis X]-Sp 34 Neu5Ac-α-2,3-Gal-β-1,3-(Fuc-α-1,4)-GlcNAc-β-[Sialyl Lewis A]-Sp 35 Neu5Gc-α-2,3-Gal-β-1,3-(Fuc-α-1,4)-GlcNAc-β-[Sialyl Lewis A]-Sp 37 Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,4-Glc-β-[LNnT]-Sp 38 GlcA-β-1,4-GlcNAc-α-1,4-GlcA-β-Sp 39 GlcNAc-β-1,6-(Gal-β-1,3)-GalNAc-α-O-Ser-Sp4 40 Neu5Ac-α-2,3Gal-β-1,4-(6S)GlcNAc-β-Sp 41 GalNAc-β-1,4-GlcNAc-β-Sp2 42 Neu5Ac-α-2,8-Neu5Ac-α-2,3-Gal β-1,4-Glc-β-Sp 43 Neu5Gc-α-2,8-Neu5Ac-α-2,3-Gal-β-1,4-Glc-β-Sp 44 GalNAc-α-1,3-(Fuc-α-1,2)-Gal-β-1,4-Glc-β-[Blood A antigen tetrose]-Sp1 45 GlcNAc-β-1,2-Man-α-Sp 46 Neu5Ac-α-2,3-Gal-β-Sp1 47 Gal-β-1,3-GalNAc-β-1,3-Gal-β-Sp1 48 Glc-α-1,2-Gal-α-Sp 49 Gal-β-1,4-(Fuc-α-1,3)-GlcNAc-β-1,3-Gal-β-Sp1 50 Neu5Ac-α-2,3-Gal-β-1,4-(Fuc-α-1,3)-Glc-β-[3-Sialyl-3- fucosyllactose/F-SL]-Sp1 51 GlcNAc-β-1,4-GlcNAc-β-Sp1 52 β-D-GlcA-Sp 53 Gal-β-1,4-(6S)GlcNAc-β-Sp 54 GlcNAc-α-1,3-(Glc-α-1,2-Glc-α-1,2)-Gal-α-1,3-Glc-α-Sp 55 Gal-β-1,3-GalNAc-β-1,4-(Neu5Gc-α-2,3)-Gal-β-1,4-Glc-β-Sp1 56 Sisomicin Sulfate 57 GalNAc-α-1,3-(Fuc-α-1,2)-Gal-β-[Blood A antigen trisaccharide]-Sp1 58 Fuc-α-1,2-Gal-β-1,4-GlcNAc-β-[Blood H antigen trisaccharide]-Sp1 60 Fuc-α-1,2-Gal-β-1,3-GlcNAc-β-1,3-Gal-β-1,4-Glc-β-[LNFP I]- Sp1 61 Fuc-α-1,2-Gal-β-1,4-Glc-β-[Blood H antigen trisaccharide]-Sp1 63 (Fuc-α-1,2)-Gal-β-1,4-(Fuc-α-1,3)-GlcNAc-β-[Lewis Y]-Sp1 64 (Fuc-α-1,2)-Gal-β-1,3-(Fuc-α-1,4)-GlcNAc-β-[Lewis B]-Sp1 65 Gal-β-1,3-(Fuc-α-1,4)-GlcNAc-β-1,3-Gal-β-1,4-(Fuc-α-1,4)- Glc-β-[Lewis A]-Sp1 66 Gal-β-1,3-GalNAc-β-Sp1 67 Gal-β-1,3-(Neu5Ac-α-2,6)-GalNAc-β-Sp 68 Neu5Ac-α-2,6-Gal-β-1,3-GalNAc-β-Sp 69 Neu5Ac-α-2,6-Gal-β-1,3-(Neu5Ac-α-2,6)-GalNAc-β-Sp 70 Neu5Ac-α-2,3-Gal-β-1,3-(Neu5Ac-α-2,6)-GalNAc-β-Sp 71 Neu5Ac-α-2,6-(Neu5Ac-α-2,3)-Gal-β-1,3-GalNAc-β-Sp 72 GalNAc-β-1,4-(Neu5Ac-α-2,3)-Gal-β-1,4-Glc-β-[GM2]-Sp 73 GalNAc-β-1,4-(Neu5Ac-α-2,8-Neu5Ac-α-2,3)-Gal-β-1,4-Glc- β-[GD2]-Sp 75 β-D-Rha-Sp 76 Glc-α-1,4-Glc-β-Sp1 77 Glc-α-1,6-Glc-α-1,4-Glc-β-Sp1 78 Maltotriose-β-Sp1 79 Glc-α-1,6-Glc-α-1,6-Glc-β-Sp1 80 Maltotetraose-β-Sp1 81 GlcNAc-α-1,4-GlcA-β-1,4-GlcNAc-α1,4-GlcA-β-Sp 82 Maltohexaose-β-Sp1 83 Maltoheptaose-β-Sp1 84 Acarbose-β-Sp1 85 D-pentamannuronic acid-β-Sp1 86 L-pentaguluronic acid-β-Sp1 87 D-cellose-β-Sp1 89 β-1,4-Xylotetrose-Sp1 90 Chitin-trisaccharide-Sp1 91 KDN-α-2,8-Neu5Ac-α-2,3-Gal-β-1,4-Glc-β-Sp 92 Neu5Ac-α-2,8-Neu5Gc-α-2,3-Gal-β-1,4-Glc-β-Sp 93 Neu5Ac-α-2,8-Neu5Ac-α-2,8-Neu5Ac-α-2,3-Gal-β-1,4-Glc-β- Sp3 94 Neu5Ac-a-2,8-Neu5Ac-α-2,6-Gal-b-1,4-Glc-Sp5 95 Gal-β-1,3-GalNAc-β-1,4-(Neu5Ac-α-2,3)-Gal-β-1,4-Glc-β-Sp1 96 Gentamicin Sulfate 97 Kanamycin sulfate 98 Geneticin Disulfate Salt (G418) 99 Neomycin trisulfate 100 SGP Linkers: Sp: OCH₂CH₂CH₂NH₂ Sp1: NH(CH₃)OCH₂CH₂NH₂ Sp2: OCH₂CH₂NH₂ Sp3: O(CH₂)₃NHCOCH₂(OCH₂CH₂)₅CH₂CH₂NH₂ Sp4: OCH₂CH(COOH)NH₂ Sp5: NH₂(-o-phenyl)-CONH-CH₂CH₂NH₂

In the presence of L-rhamnose, SHA/rSHA binding to all glycans, except α-Rha, was competitively inhibited (FIGS. 6A and 6B). These results are consistent with earlier hemagglutination inhibition or equilibrium dialysis observations in which SHA bound to L-rhamnose with a higher affinity than to D-galactose (2-4).

Example 7: Sequence Comparison of SHA and Putative Homologues

SHA homologues were identified not only in the Streptomyces genome, but also in the genomes of other microorganisms. Eleven putative SHA homologues with more than 50% homology to the SHA sequence were identified as N-terminally truncated hypothetical proteins in the genomes of S. lavendulae, S. sp. Mg1, S. sp. Wm4235, S. xanthophaeus, S. sp. Wm6378, S. clavuligerus, S. scabiei, Streptacidiphilus melanogenes, Lentzea sp. DHS C013, Actinobacteria bacterium, and Nocardia sp. NRRL S-836 (FIG. 8A). The N-terminal sequence of the putative SHA homologues varied among homologues, and a corresponding sequence was absent in SHA. In contrast, the C-terminal domain was conserved between SHA and its homologues. Compared to the 131 amino acids of the SHA sequence, the SHA homologues contained 15-133 additional amino acids at the N-terminal end, for a total of 172-265 amino acids.

To compare protein and DNA sequences of SHA and its homologues, a phylogenetic tree was generated (FIG. 8B). Protein sequence homology ranged from 51-99%. In the absence of SHA genetic information, S. lavendulae DNA (438 bases) was used as the reference query for SHA homologues. DNA sequence homology ranged from 67-82%.

The primary sequence of SHA is principally made up of three homologous “SHA domains,” each consisting of 29 to 33 amino acids (Table 2). Sequence identity between the three SHA domains ranged from 60% to 70% (FIG. 8C). The three SHA domains contained an identical stretch of eleven consecutive amino acids, GTTGQSRRMEA (SEQ ID NO: 24), at the C-terminus. Together, they comprised 92 amino acids, 70% of the total 131 amino acids in SHA. Furthermore, the SHA domains showed homology to tryptophan-rich ChW domains. ChW domains are almost exclusively found in the Clostridium acetobutylicum species (9,10). Protein Q97E41 of Clostridium acetobutylicum ATCC 824 was identified as the closest clostridial homologue to SHA, using SMART (simple modular architecture research tool). It had 59%, 39%, and 37% identity for SHA domains 1, 2, and 3, respectively (Table 3).

Example 8: NMR Identification of Tryptophan Residues

NMR titration was used to show that the addition of L-rhamnose caused chemical shifts in NMR signals from SHA in the tryptophan indole NH and methyl group regions (FIG. 9). This indicates that the ChW tryptophan residues are most likely directly involved in carbohydrate binding.

Example 9: Demonstration of rSHA Binding to Microbial Surfaces

Due to the loss of the original SHA-producing Streptomyces strain 27S5, the biological role of SHA is difficult to characterize. SHA binding to microbial cell surfaces was demonstrated in this example. A green fluorescent protein (GFP) SHA fusion protein (GFP-SHA) was constructed and used to stain various bacteria and fungi, and performed fluorescence microscopy. A representative example is shown in FIG. 10, which demonstrates the binding of GFP-SHA to Lactobacillus casei Shirota cells. L. casei Shirota is rich in L-rhamnose-containing cell wall glycans (11). The binding of SHA to microbial glycans may imply a role for SHA in complex microbial communication.

Example 10: ⁶⁸Ga PET Imaging of CD-1 Mice

PET imaging was performed using with 68-gallium labeled GFP-SHA fusion protein in mice. ⁶⁸Ga was obtained from a ⁶⁸Ge/⁶⁸Ga generator system and chelated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-labeled GFP-SHA. 5.1 MBq of the resulting ⁶⁸Ga-GFP-SHA was injected through the tail vein of female CD1 mice, and PET imaging was conducted at the City of Hope Small Animal Imaging Core facility. As a control ⁶⁸Ga-DOTA-scFv was used. The PET scanning was conducted 1.5 hours after intravenous injection of the PET imaging agent. The ⁶⁸Ga-DOTA-scFv had no specificity for rhamnose-containing microorganisms. As shown in FIG. 11, ⁶⁸Ga-labeled GFP-SHA specifically labels parts of the small intestine and the cecum. These are the areas that contain microorganisms.

While the control mouse only shows radioactivity uptake in the kidneys and in the bladder, which is typical for small proteins, the ⁶⁸Ga-DOTA-GFP-SHA-injected mouse reveals additional strong signals from the cecum and the small intestine. The latter are known internal organs that naturally contain a rhamnose-rich microbial flora.

Example 11: Staining of LS180 Cells with GFP-SHA

LS180 human colon adenocarcinoma cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) with 4.5 g/L glucose, L-glutamine, and sodium pyruvate (Corning) with seeding at 60,000 cells/well in a 24-well dish containing glass coverslips. Cells were adhered overnight, after which media was removed and coverslips washed 3× with phosphate-buffered saline (PBS). Paraformaldehyde (4%) was then added with incubation for 15 min at 25° C., followed by washing 3× with PBS and blocking with 5% bovine serum albumin (BSA) in PBS for one hour. Cells were then incubated with 10 μg/mL GFP-SHA or a GFP-only control diluted in 5% BSA in PBS overnight at 4° C. Following staining, cells were washed 3× with PBS and then mounted on glass slides with 10 μL Fluoroshield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, Abcam). Cells were then visualized using a Zeiss LSM 880 with Airyscan confocal microscope, employing excitation at 488 nm for green fluorescence and 358 nm for DAPI staining. As shown in FIG. 11, GFP-SHA binds to the surface of LS180 cells, while GFP alone does not.

Example 12: SHA Imaging of Cancer Tissues Using FITC-Labeled SHA

SHA was labeled with FITC using amine coupling chemistry. Briefly, 100 μL of SHA in 0.1 M sodium carbonate, pH 9.0 at 2 mg/mL concentration was incubated for 1 hour with 50 μg/mL of FITC in DMSO on rotary shaker at 22° C. (protected from light). After incubation, the reaction was quenched by adding 20 μL of 1 M ethanolamine following 30 minutes incubation at 22° C. Purification of labeled protein was performed using 5 kDa MW cut off filter. The degree of labeling at 1.7 (Fluorophore/Protein) was calculated using 68,000 cm⁻¹M⁻¹ molar extinction coefficient of the dye at pH 8.0 at 494 nm.

7 μm thick consecutive sections from each block of a paraffinized formalin fixed tissue sample were cut and baked, and each section was placed on a separate slide. Sections were deparaffinized using sequential immersions into 2 xylene baths (10 minutes each), dehydrated with 4 baths of decreasing alcohol concentrations (100%, 95%, 70%, and 50%, 5 minutes each) and 2 baths with deionized H₂O. Slides then were rehydrated with 1×PBS for 10 minutes following heat-induced antigen unmasking procedure. Briefly, slides for 8 minutes at 95° C. in 10 mM citrate buffer, pH 6.0, rinsed gently with deionized H₂O and then with 1×PBS. Staining procedure began with blocking of slides overnight at 4° C. with 1×PBS with 5% bovine serum albumin following incubation with FITC-SHA at 20 μg/mL for 1 hour at 22° C. in blocking buffer. Slides were washed three times with 1×PBS. Counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI, 3 μM for 10 minutes, washed with 1×PBS three times and examined using a fluorescent microscope. Hematoxylin and Eosin (H&E) staining was performed using standard hematoxylin and eosin staining procedure (www.nationaldiagnostics.com). Microscopy was performed with Zeiss Axio Observer II Inverted Fluorescence Microscope (Jena, Germany) and ZEN2 (Blue Edition) software.

The FITC-SHA-stained tissues of the infiltrating ductal carcinoma case (which was an infiltrating, malignant and abnormal proliferation of neoplastic cells in the breast tissues advanced to the tumor in the pancreas) were examined. As shown in FIG. 13, the SHA clearly labeled cells in tumor tissues. Pancreatic ductal carcinoma gave very strong staining. Other tumors did too (data not shown). The precise target that was SHA-labeled in the tumor sections is unknown at this point, but not wishing to be bound by any theory, it might be due to abnormal mucins secreted by tumor cells.

Significant difference in fluorescent signal was observed between the two groups, the pancreatic cancer tissue and the normal tissue stained with FITC-SHA protein demonstrating that the FITC-SHA has higher affinity to cancerous cells than to the normal cells.

As stated above, the foregoing are merely intended to illustrate the various embodiments of the present invention. As such, the specific modifications discussed above are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein. All references cited herein are incorporated by reference as if fully set forth herein.

REFERENCES

-   1. Fujita, Y., Oishi, K., and Aida, K. (1972) Hemagglutination by     culture broth of Actinomycetes and Aspergillus. J. Gen. Appl.     Microbiol. 18, 73-75 -   2. Fujita, Y., Oishi, K., and Aida, K. (1973) Sugar specificity of     anti-B hemagglutinin produced by Streptomyces sp. Biochemical and     biophysical research communications 53, 495-501 -   3. Fujita, Y., Oishi, K., Suzuki, K., and Imahori, K. (1975)     Purification and properties of an anti-B hemagglutinin produced by     Streptomyces sp. Biochemistry 14, 4465-4470 -   4. Fujita-Yamaguchi, Y., Oishi, K., Suzuki, K., and     Imahori, K. (1982) Studies on carbohydrate binding to a lectin     purified from Streptomyces sp. Biochimica et biophysica acta 701,     86-92 -   5. Harrison, J., and Studholme, D. J. (2014) Recently published     Streptomyces genome sequences. Microbial biotechnology 7, 373-380 -   6. Hoefler, B. C., Konganti, K., and Straight, P. D. (2013) De Novo     Assembly of the Streptomyces sp. Strain Mg1 Genome Using PacBio     Single-Molecule Sequencing. Genome announcements 1 -   7. Fujita, Y. (1976) Studies on hemagglutinins produced by     microorganisms. The University of Tokyo -   8. Guerrero, F., Ciragan, A., and Iwai, H. (2015) Tandem SUMO fusion     vectors for improving soluble protein expression and purification.     Protein expression and purification 116, 42-49 -   9. Nolling, J., Breton, G., Omelchenko, M. V., Makarova, K. S.,     Zeng, Q., Gibson, R., Lee, H. M., Dubois, J., Qiu, D., Hitti, J.,     Wolf, Y. I., Tatusov, R. L., Sabathe, F., Doucette-Stamm, L.,     Soucaille, P., Daly, M. J., Bennett, G. N., Koonin, E. V., and     Smith, D. R. (2001) Genome sequence and comparative analysis of the     solvent-producing bacterium Clostridium acetobutylicum. Journal of     bacteriology 183, 4823-4838 -   10. Sullivan, L., Paredes, C. J., Papoutsakis, E. T., and     Bennett, G. N. (2007) Analysis of the clostridial hydrophobic with a     conserved tryptophan family (ChW) of proteins in Clostridium     acetobutylicum with emphasis on ChW14 and ChW16/17. Enzyme and     Microbial Technology 42, 29-43 -   11. Yasuda, E., Tateno, H., Hirabayashi, J., Iino, T., and     Sako, T. (2011) Lectin microarray reveals binding profiles of     Lactobacillus casei strains in a comprehensive analysis of bacterial     cell wall polysaccharides. Applied and environmental microbiology     77, 4539-4546 -   12. Zhang, Q., Ames, J. M., Smith, R. D., Baynes, J. W., and     Metz, T. O. (2009) A perspective on the Maillard reaction and the     analysis of protein glycation by mass spectrometry: probing the     pathogenesis of chronic disease. Journal of proteome research 8,     754-769 -   13. Negre-Salvayre, A., Salvayre, R., Auge, N., Pamplona, R., and     Portero-Otin, M. (2009) Hyperglycemia and glycation in diabetic     complications. Antioxidants & redox signaling 11, 3071-3109 -   14. Cheng, H. N., and Neiss, T. G. (2012) Solution NMR spectroscopy     of food polysaccharides. Polymer Reviews 52, 81-114 -   15. Fujimoto, Z., Jackson, A., Michikawa, M., Maehara, T., Momma,     M., Henrissat, B., Gilbert, H. J., and Kaneko, S. (2013) The     Structure of a Streptomyces avermitilis α-I-Rhamnosidase Reveals a     Novel Carbohydrate-binding Module CBM67 within the Six-domain     Arrangement. The Journal of Biological Chemistry 288, 12376-12385 -   16. Lamoth, F. (2016) Galactomannan and 1,3-β-D-Glucan Testing for     the Diagnosis of Invasive Aspergillosis. J. Fungi 2, 22 -   17. Tateno, H., Saneyoshi, A., Ogawa, T., Muramoto, K., Kamiya, H.,     and Saneyoshi, M. (1998) Isolation and characterization of     rhamnose-binding lectins from eggs of steelhead trout (Oncorhynchus     mykiss) homologous to low density lipoprotein receptor superfamily.     The Journal of biological chemistry 273, 19190-19197 -   18. Hosono, M., Ishikawa, K., Mineki, R., Murayama, K., Numata, C.,     Ogawa, Y., Takayanagi, Y., and Nitta, K. (1999) Tandem repeat     structure of rhamnose-binding lectin from catfish (Silurus asotus)     eggs. Biochimica et biophysica acta 1472, 668-675 -   19. Tateno, H., Ogawa, T., Muramoto, K., Kamiya, H., and     Saneyoshi, M. (2002) Distribution and molecular evolution of     rhamnose-binding lectins in Salmonidae: isolation and     characterization of two lectins from white-spotted Charr (Salvelinus     leucomaenis) eggs. Bioscience, biotechnology, and biochemistry 66,     1356-1365 -   20. Tateno, H., Ogawa, T., Muramoto, K., Kamiya, H., and     Saneyoshi, M. (2002) Rhamnose-binding lectins from steelhead trout     (Oncorhynchus mykiss) eggs recognize bacterial lipopolysaccharides     and lipoteichoic acid. Bioscience, biotechnology, and biochemistry     66, 604-612 -   21. Terada, T., Watanabe, Y., Tateno, H., Naganuma, T., Ogawa, T.,     Muramoto, K., and Kamiya, H. (2007) Structural characterization of a     rhamnose-binding glycoprotein (lectin) from Spanish mackerel     (Scomberomorous niphonius) eggs. Biochimica et biophysica acta 1770,     617-629 -   22. Tateno, H. (2010) SUEL-related lectins, a lectin family widely     distributed throughout organisms. Bioscience, biotechnology, and     biochemistry 74, 1141-1144 -   23. Ogawa, T., Watanabe, M., Naganuma, T., and Muramoto, K. (2011)     Diversified carbohydrate-binding lectins from marine resources.     Journal of amino acids 2011, 838914 -   24. Ozeki, Y., Yokota, Y., Kato, K. H., Titani, K., and     Matsui, T. (1995) Developmental expression of D-galactoside-binding     lectin in sea urchin (Anthocidaris crassispina) eggs. Experimental     cell research 216, 318-324 -   25. Watanabe, Y., Tateno, H., Nakamura-Tsuruta, S., Kominami, J.,     Hirabayashi, J., Nakamura, O., Watanabe, T., Kamiya, H., Naganuma,     T., Ogawa, T., Naude, R. J., and Muramoto, K. (2009) The function of     rhamnose-binding lectin in innate immunity by restricted binding to     Gb3. Developmental and comparative immunology 33, 187-197 -   26. Sharon, N. (1987) Bacterial lectins, cell-cell recognition and     infectious disease. FEBS letters 217, 145-157 -   27. Ng, S. K., Huang, Y. T., Lee, Y. C., Low, E. L., Chiu, C. H.,     Chen, S. L., Mao, L. C., and Chang, M. D. (2014) A recombinant     horseshoe crab plasma lectin recognizes specific pathogen-associated     molecular patterns of bacteria through rhamnose. PloS one 9, el     15296 -   28. Carpentier, G. (2010) Contribution: Protein Array Analyzer for     ImageJ. ImageJ News 10, presented at the poster session at the     ImageJ User and Developer Conference, Oct. 27-29, 2010 -   29. Suzanne V. Smith, Marian Jones and Vanessa Holmes (2011).     Production and Selection of Metal PET Radioisotopes for Molecular     Imaging, Radioisotopes—Applications in Bio-Medical Science, Prof.     Nirmal Singh (Ed.), ISBN: 978-953-307-748-2 

1.-27. (canceled)
 28. A method of detecting a microbial infection caused by a microorganism expressing a carbohydrate containing L-rhamnose or D-galactose on the surface in a subject, comprising: contacting a sample obtained from the subject with a fusion protein comprising a fluorescent protein and SHA or a homologue of SHA, wherein the SHA protein has an amino acid sequence represented by SEQ ID NO: 25, the homologue has an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to that of SEQ ID NO: 25, and wherein the fusion protein specifically binds to L-rhamnose or D-galactose; and detecting the fluorescence level in the sample, wherein the presence of fluorescence indicating a microbial infection.
 29. The method of claim 28, wherein the sample includes a biopsy sample, a tissue sample, a bronchoalveolar lavage sample, a blood sample, and a urine sample.
 30. The method of claim 28, wherein the microbial infection is caused by a bacterium or a fungus that expresses dTDP-4-dehydrorhamnose reductase gene (rmID).
 31. The method of claim 28, wherein the microbial infection includes mycoses caused by Candida albicans, Aspergillus fumigatus, or Fusarium solani.
 32. The method of claim 28, wherein the microbial infection is an infection by Streptococcus, Enterococcus or Lactococcus.
 33. The method of claim 28, wherein the microbial infection is invasive pulmonary aspergillosis, and the GFP-SHA fusion protein detects the presence of fungal galactomannan.
 34. The method of claim 28, wherein the fusion protein specifically binds to a carbohydrate that contains or terminates in a monosaccharide or an oligosaccharide selected from the group consisting of β-Gal-; α-Rha-; Gal-α-1,3-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-Gal-β-1,4-Glc-β-; Gal-α-1,4-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-(Fuc-α-1,2)-Gal-β-; Gal-α-1,3-(Fuc-α-1,2)-Gal-β-1,4-Glc-β-; Gal-α-1,3-Gal-β-, Gal-α-1,4-Gal-β-1,4-Glc-β-; GalNAc-β-1,3-Gal-β-1,4-Glc-β-; and Gal-α-1,4-Gal-β-1,4-GlcNAc-β-.
 35. The method of claim 28, wherein the fluorescent protein includes GFP and mCherry1.
 36. A method of detecting a cancer or tumor in a subject, comprising: contacting a sample obtained from the subject with a fusion protein comprising a fluorescent protein and SHA or a homologue of SHA, wherein the SHA protein has an amino acid sequence represented by SEQ ID NO: 25, the homologue has an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to that of SEQ ID NO: 25, and wherein the fusion protein specifically binds to L-rhamnose or D-galactose; and detecting the fluorescence level in the sample, wherein the presence of fluorescence indicating the presence of the cancer or tumor, wherein the cancer or tumor cell expresses an antigen comprising a carbohydrate capable of specifically binding to SHA, a homologue thereof, or a fragment of the SHA or a homologue thereof.
 37. The method of claim 36, wherein the sample includes a biopsy sample, a tissue sample, a bronchoalveolar lavage sample, a blood sample, and a urine sample.
 38. The method of claim 36, wherein the cancer is colon cancer, pancreatic ductal carcinoma, or pancreatic cancer.
 39. The method of claim 36, wherein the antigen is a surface antigen comprising a carbohydrate containing D-galactose.
 40. The method of claim 36, wherein the fusion protein specifically binds to a carbohydrate that contains or terminates in a monosaccharide or an oligosaccharide selected from the group consisting of β-Gal-; α-Rha-; Gal-α-1,3-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-Gal-β-1,4-Glc-β-; Gal-α-1,4-Gal-β-1,3-GlcNAc-β-; Gal-α-1,3-(Fuc-α-1,2)-Gal-β-; Gal-α-1,3-(Fuc-α-1,2)-Gal-β-1,4-Glc-β-; Gal-α-1,3-Gal-β-, Gal-α-1,4-Gal-β-1,4-Glc-β-; GalNAc-β-1,3-Gal-β-1,4-Glc-β-; and Gal-α-1,4-Gal-β-1,4-GlcNAc-β-.
 41. The method of claim 36, wherein the fluorescent protein includes GFP and mCherry1.
 42. A method of imaging an organ or tissue having a microbial infection caused by a microorganism expressing a carbohydrate containing L-rhamnose or D-galactose on the surface in a subject, comprising: administering to the subject a positron emission tomography (PET) probe comprising a recombinant SHA protein or the homologue thereof labeled with a PET isotope, or a fusion protein labeled with a PET isotope; and imaging the organ or the tissue having the microbial infection by a PET scanning of the subject, wherein the fusion protein comprises a fluorescent protein and SHA or a homologue of SHA, wherein the SHA protein has an amino acid sequence represented by SEQ ID NO: 25, the homologue has an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to that of SEQ ID NO: 25, and wherein the fusion protein specifically binds to L-rhamnose or D-galactose.
 43. The method of claim 42, wherein the PET probe is administered locally or systemically to the organ or the tissue.
 44. The method of claim 42, wherein the PET probe is administered by intravenous injection.
 45. A method of imaging a solid tumor in a subject, comprising: administering to the subject a positron emission tomography (PET) probe comprising a recombinant SHA protein or the homologue thereof labeled with a PET isotope, or a fusion protein labeled with a PET isotope, and imaging the solid tumor by a PET scanning of the subject, wherein the tumor cell expressing a tumor-specific antigen comprising a carbohydrate capable of specifically binding to SHA, a homologue thereof, or a fragment of the SHA or a homologue thereof, wherein the fusion protein comprises a fluorescent protein and SHA or a homologue of SHA, wherein the SHA protein has an amino acid sequence represented by SEQ ID NO: 25, the homologue has an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to that of SEQ ID NO: 25, and wherein the fusion protein specifically binds to L-rhamnose or D-galactose.
 46. The method of claim 45, wherein the PET probe is administered locally to the tumor.
 47. The method of claim 45, wherein the antigen is a surface antigen comprising a carbohydrate containing D-galactose. 